Die dreidimensionale Struktur des Influenzavirus-Hämagglutinin im membranfusionsaktiven Zustand

by Ludwig, Kai

Abstract Envelope viruses enter a host cell via fusion between the viral and endosomal membrane, thereby releasing the nucleocapsid into the cytoplasm. The fusion has been accompanied with an conformational change in the ectodomain of viral glycoproteins. To understand the mechanism leading to fusion the three-dimensional (3D) structure of the complete viral glycoprotein in its fusion competent conformation has to be determined. The Fusion of influenza virus which is triggered at low pH has been associated with an irreversible conformational change in the trimeric glycoprotein hemagglutinin (HA). The three-dimensional (crystal) structure is already known of the enzymatic-cleaved ectodomain of one influenza strain (X-31) at neutral pH or of some fragments of the HA2-subunit at low pH, respectively. In this work a suitable influenza strain as well as suitable experimental conditions for investigations of the 3D-structure of the complete and not enzymatically treated HA at neutral and acidic pH conditions have been determined. An cryo-microscopy/angular reconstitution approach has been employed for the 3D- reconstruction of the intact HA of an different influenza strain (A/Japan). This structure is in excellent agreement with the known X-ray crystallographic structure of the bromelain- cleaved ectodomain of HA from influenza X-31. Moreover, for the very first time the 3D structure of the intact HA of Influenza A/Japan in its fusion competent state (at acidic pH) has been calculated. The differences between the two structures are large compared to the marginal differences between the neutral pH structures by EM and by X-ray crystallography, respectively, although the monomers remain tightly connected. These findings will be discussed especially with regard to the role of the HA1 subunit in the fusion process. The procedure is in general applicable to pursue the 3D-structure of (fusion mediating) proteins in various conformational states (especially of those proteins which are not directly accessible by X-ray crystallography). This approach should offer to elucidate the (eventually conserved) mechanism of membrane fusion.